This is an application for an ABI-Sciex 5500 Qtrap to support research by 8 named NIH investigators who are carrying out research on biological pathways, protein complexes, protein post-translational modifications and small biologically active peptides in living systems. The 5500 Qtrap has significant sensitivity advantages over the existing 4000 Qtrap with a 5-fold increase in the multiple reaction ion monitoring (MRM) mode and >30 fold for MSMS analysis. The high sensitivity plus the very fast scanning speed (up to 20,000 m/z per sec) is a significant advance. It allows the monitoring of 100 MRM channels with the ability to capture a confirmatory MSMS spectrum during the passage of a peak in each MRM channel. With elution time scheduling, the number of channels monitored in a single LC run can be substantially increased. This instrument is well suited to directed proteomics in which the proteins of interest are well-defined, either by other protein mass spectrometry experiments, from DNA microarray analysis, gene sequencing, or from other biological results. In this approach, peptides representing proteins are carefully selected based on length (8-18 residues), amino acid composition (omitting peptides with easily oxidized residues - Cys, His, Met, Trp and Tyr) and sequence uniqueness to act as surrogates. These peptides can also be measured quantitatively by the preparation of stable isotope-labeled forms. This allows whole pathways of proteins/enzymes or protein complexes to be analyzed in a single LC run and hence to understand the role of protein turnover. These include the proteasome complex in activated neutrophils, the enzymes in glycolysis and energy generating mitochondrial pathways, and the lens crystallins in aging. The large number of MRM channels also allows the study of heavily modified proteins such as the cystic fibrosis transmembrane regulator (we have observed over 60 post- translational modifications) and lens crystallins. The high sensitivity of the 5500 Qtrap will also allow measurement of small peptides such as those produced from collagen in the lung in chronic obstructive pulmonary disease to be measured in more convenient/accessible fluids such plasma or urine. The key issue in all these experiments is that the investigators want to carry out analyses on proteins in biological systems at normal abundance levels. The increased sensitivity of the 5500 Qtrap significantly improves that capability. PUBLIC HEALTH RELEVANCE: The requested instrument is much more sensitive than its predecessors and will allow investigators to examine the complexity of protein chemistry in living tissues. It will be applied to the study of mechanisms of neutrophilassociated damage in the lung, mitochondrial-associated damage in models of oxidative stress (atherosclerosis, diabetes and cancer), lens cataract disease in aging, pathway robustness in the light of gene damage/loss, and in Cystic Fibrosis. It's very well suited to obtaining quantitative data to better understand the role of the protein(s) in specific areas of public health.